Diabetes
Insulin is a hormone produced by the pancreas that moves sugar from the bloodstream into the cells of the body, where it becomes an essential energy source. In mammals, insulin is synthesized in the pancreas within the beta cells (3-cells) of the islets of Langerhans (pancreatic islets). There are about one million islets in a healthy adult human pancreas (about 1-2% of the total mass of the pancreas), which are distributed throughout the organ
Diabetes is a disease state characterized by abnormally high levels of sugar (hyperglycemia) in the blood, either because the body does not produce enough insulin (Type 1 diabetes) or because the body cannot respond to the insulin that is produced (Type 2 diabetes). Uncontrolled, hyperglycemia can lead to serious complications including blindness, heart disease, kidney disease and even death.
In the United States alone, more than 20 million people have diabetes. Type 2 diabetes (T2D) is by far the most common type, and is associated with lack of physical activity and obesity. According to statistics compiled by the World Health Organization (WHO), in 2007, over 180 million people world wide have diabetes, resulting in 2.9 million deaths (6% of total global mortality), and associated with a total economic burden of more than $230 billion.
Type 1 diabetes (T1D) is far less common than T2D. It is an autoimmune disease in which the patient's own immune system destroys the body's insulin-producing pancreatic beta cells. Typically diagnosed at a young age, it is a chronic disease that requiring life-long treatment. Treatment is generally in the form of insulin replacement therapy, which is typically delivered by injection or pump. Successful insulin management depends on how closely a given regimen can mimic normal physiologic insulin release patterns. There are several different forms of insulin available, and the choice of a particular form/regime may reflect that patient's preferences and ability to adhere to a particular treatment regime. Despite advances in the pharmacology and delivery of insulin, achieving tight glycemic control using insulin replacement therapy can be very demanding. As a result, many T1D patients still experience episodes of hyper- and hypo-glycemia and suffer long term complications as a result.
Given the burdens of insulin replacement therapy, therapeutic alternatives are highly desirable. Transplanted human pancreases (allografts) offer a potential cure for T1D patients. Sources include human donors that have recently deceased or living donors (partial pancreas transplant). The recipient's native pancreas is generally left in place, and the donated pancreas is attached in a different location. Challenges include the risks inherent in any surgical procedure as well the possibility of rejection common to most transplanted organs. Rejection of the allograft pancreas may occur at any time from within seconds (acute) to years (chronic) after transplantation. To avoid rejection, immunosuppressive drugs must be taken indefinitely. These drugs can be difficult to tolerate, leave the patient at increased risk for infectious disease and have also been linked to high blood pressure, kidney problems and liver disorders. The risks of transplantation and the extended use of immunosuppressive drug therapy are uniquely problematic for diabetic patients (i.e., compared to other organ transplant recipients), as drug therapy generally remains an option, however undesirable. A 2003 study found that for patients with functioning kidneys, survival rates of patients who receive pancreas-only transplants were worse than the survival rates of patients who manage their diabetes with conventional therapy (Venstrom et al. 2003; 290:2817-2823). As a result, pancreas transplantation is normally only performed on individuals with type 1 diabetes with end-stage renal disease.
Transplant of only the islet cells (versus the entire pancreas) provides a less invasive transplant-based alternative. Here, islets are isolated from the donor pancreas and injected into the patient via a catheter to the portal vein (i.e., no requirement for a major abdominal incision). The islets travel to the liver where they become fixed, taking over insulin production and essentially turning the liver into a replacement pancreas. Early islet transplants had very low success rates, however, and patients remained insulin-independent for only short periods of time. The major differences between the Edmonton Protocol and those early islet transplant procedures was the use of a particular combination of immunosuppressive drugs and transplant of islets from more than one pancreas. Specifically, the Edmonton protocol uses a combination of immunosuppressive drugs that includes daclizumab, sirolimus and tacrolimus Daclizumab is given intravenously immediate post-transplant and then discontinued. The patient is then given sirolimus and tacrolimus indefinitely.
Both whole pancreas and islet transplant procedures depend on a reliable supply of human pancreas donors, which doesn't currently exist. At present, only 3000 cadaver pancreases become available each year, far short of those needed for the 2 million plus patients with T1D.
Gene therapy presents another therapeutic alternative. The introduction and expression of transgenes in human pancreatic islets to prevent immune rejection and improve proliferation and survival of islet grafts has been the focus of much research (review by McCabe et al., Diabetes Metab Res Rev. 2006 May-June; 22(3):241-52; Chuang et al., 2008; Martin et al., Endocr Dev. 2007; 12:24-32; Faideau et al., Diabetes. 2005 December; 54 Suppl 2:S87-96). Transgene delivery via ex-vivo transduction of human islets has been investigated (Garcia-Ocana et al., Journal of Biol Chem., 2003, 278:343-351; Li et al., Transplantation Proceedings, 39:3436-3437). However, the immunomodulatory gene expression in these systems was insufficient for long term diabetic control as adenovirally infected islet grafts were rejected in about one month (see Sakata et al., Diabetes Research and Clinical Practice, 2008, 80:352-359). In addition, adenoviral vectors used for gene therapy in humans are limited in their capacity to deliver certain genes and have triggered immune responses and even caused one death (Flotte, J. of Cellular Physiology, 2007, 213:301-305). The efficiency of alternative, non-viral gene delivery systems has been low and transient. Genetic modification of human pancreatic cells has therefore failed to effectively address the needs of T1D patients.
Xenotransplantation
Xenotransplantation (transplant of organs, tissues and cells from a donor of a different species) could effectively address the shortage of human donor pancreases. Xenotransplants are also advantageously (i) supplied on a predictable, non-emergency basis; (ii) produced in a controlled environment; and (iii) available for characterization and study prior to transplant.
Depending on the relationship between donor and recipient species, the xenotransplant can be described as concordant or discordant. Concordant species are phylogenetically closely related species (e.g., mouse to rat). Discordant species are not closely related (e.g., pig to human). Pigs have been the focus of most research in the xenotransplanation area, since the pig shares many anatomical and physiological characteristics with human. Pigs also have relatively short gestation periods, can be bred in pathogen-free environments and may not present the same ethical issues associated with animals not commonly used as food sources (e.g., primates).
Scientific knowledge and expertise in the field of pig-to-primate xenotransplantation has grown rapidly over the last decade, resulting in the considerably prolonged survival of primate recipients of lifesaving porcine xenografts. (Cozzi et al., Xenotransplantation, 16:203-214. 2009). Recently, significant achievements have been reported in the field of islet xenotransplantation (Hering B J, et al., Nat Med, 12:301-303. 2006; Cardona K, et al., Nat Med, 12:304306. 2006; Gianello P and Dufrane D., Xenotransplantation, 14: 441. 2007), and this progress has prompted to may to suggest that islets, and not solid organs, may be the first type of transplant in future clinical xenotransplantation trials.
Genetic Modification
While advantageous in many ways, xenotransplantation also creates a more complex immunological scenario than allotransplantation. As such, considerable effort has been directed at addressing the immune barrier through genetic modification (van der Windt et al., Xenotransplantation. 2007 July; 14(4):288-97, Cowan and D'Apice, Curr Opin Organ Transplant. 2008 April; 13(2):178-83).
Xenograft rejection can be divided into three phases: hyperacute rejection, acute Immoral xenograft rejection, and T cell-mediated cellular rejection. Hyperacute rejection (HAR) is a very rapid event that results in irreversible graft damage and loss within minutes to hours following graft reperfusion. It is triggered by the presence of xenoreactive natural antibodies present within the recipient at the time of transplantation. Humans have a naturally occurring antibody to the alpha 1,3-galactose (Gal) epitope found on pig cells. This antibody is produced in high quantity and, it is now believed, is the principle mediator of HAR. (Sandrin et al., Proc Natl Acad Sci USA. 1993 December 1; 90(23):11391-5, 1993; review by Sandrin and McKenzie, Immunol Rev. 1994 October; 141:169-90). Initial efforts to genetically modify pigs have focused on removing the alpha 1,3-galactose (Gal) epitope from pig cells. In 2003, Phelps et al. (Science, 2003, 299:411-414) reported the production of the first live pigs lacking any functional expression of αGT (GTKO), which represented a major breakthrough in xenotransplantation (see also PCT publication No. WO 04/028243 to Revivicor, Inc. and PCT Publication No. WO 04/016742 to Immerge Biotherapeutics, Inc.). Subsequent studies have shown that organ grafts from GTKO pigs do not undergo HAR (Kuwaki et al., Nat Med. 2005 January; 11(1):29-31, Yamada et al., Nat Med. 2005 January; 11(1):32-4). Although Gal-mediated HAR is now known to be a significant factor in xenotransplantation of whole organs.
It is not clear if HAR is also a critical factor in adult islet xenotransplantation as pure populations of pancreatic beta cells from adult pigs do not express significant levels of the immunogenic Gal epitope. Indeed, in one study, it was found that GTKO pig pancreatic islets were no less susceptible to destruction than wild type islets (Rood, et al. (2007) Transplantation 83:202-210). However, unlike adult islets, fetal and neonatal islets do express Gal.
Expression of complement regulators in xenotransplant tissue has been suggested as a different strategy to combat HAR (Squinto, Curr Opin Biotechnol. 1996 December; 7(6):641-5). European patent 0495852 to Imutran suggests associating xenograft tissues with recipient complement restriction factors to reduce complement activation in the recipient (see also Diamond, et al., Transpl Immunol. 1995 December; 3(4):305-12). Transgenic pigs expressing human DAF (hDAF) and/or human CD59 (hCD59) have been reported (Byrne et al., Transplant Proc., 1996 April; 28(2):758). CD46 has been expressed in pig cells using a minigene that was optimized for high ubiquitous expression and appears to protect porcine cells in a mouse transplantation model (Loveland et al., Xenotransplantation, 2004, 11:171:183; McKenzie et al., Xenotransplantation. 2003 November; 10(6):615-21). However, expression of these factors has been variable and generally very low in pancreatic cells (see Bennet et al., Transplantation, 2001, 72:312-319).
Even where HAR is avoided, the xenograft undergoes a delayed form of rejection, acute Immoral xenograft rejection (AHXR)—also referred to as delayed xenograft rejection (DXR). It is generally thought to be initiated by xeno-reactive antibodies, including non-Gal antibodies and subsequent activation of the graft endothelium, the complement and the coagulation systems (Miyagawa et al. Xenotransplantation, 2010, 1: 11-25).
Although the threats presented by the Immoral response are critical with regard to the survival and function of vascularized grafts, the risk of graft damage by cellular mechanisms is also important. T-cell mediated acute responses play an important role in xenotransplant rejection, although their role in transplantation of pancreatic islet cells has not been fully elucidated. Of several T cell costimulatory pathways identified to date, the most prominent is the CD28 pathway and the related cytoxic T-lymphocyte associated protein (CTLA4) pathway.
To date, much of the research on CTLA4-Ig as an immunosuppressive agent has focused on administering soluble forms of CTLA4-Ig to a patient (see U.S. Pat. No. 7,304,033; PCT Publication No. WO 99/57266; and Lui et al. J Immunol Methods 2003 277:171-183). To reduce the overall immunosuppressive burden on a patient, transgenic expression of such a protein has been suggested. Transgenic mice expressing CTLA4-Ig have been developed (Ronchese et al. J Exp Med (1994) 179:809; Lane et al. J Exp Med. (1994) March 1; 179(3):819; Sutherland et al. Transplantation. 2000 69(9):1806-12). In addition, PCT Publication No. WO 01/30966 to Alexion Pharmaceuticals, Inc. and PCT Publication No. WO 07/035213 to Revivicor discloses transgenic pigs expressing only the CTLA4-Ig transgene. See also Phelps et al., Xenotransplantation, 16(6):477-485. 2009. Pigs expressing CTLA4Ig in brain tissue were produced, but high plasma expression was shown to cause negative effects (Martin, et al. (2005) Transg. Rsch. 14:373-84). There remains doubt as to whether long term expression of immunosuppressive transgenes in ungulates raises safety concerns either for the ungulate or for the recipient of any tissues from such an animal.
In addition to the cellular and Immoral immune responses, a significant challenge associated with islet transplantation is the significant early loss of islet mass immediately after infusion of the transplanted islets and contact with recipient blood, a phenomenon known as the immediate blood-mediated inflammatory response (IBMIR) (Bennet et al., Ups J Med Sci 2000, 105:125-133). The addition of an anticoagulant transgene has been suggested to prevent coagulation responses to xenografts (reviewed by Cowan, Xenotransplantation, 2007; 14:7-12). However, these reports have focused on the reduction of coagulation associated with organ transplantation. In addition, production of anticoagulant-expressing animals suitable for xenotransplantation has proven difficult due to bleeding phenotypes seen even in small mammals such as mice (see Dwyer et al. (2004) J Clin Invest 113: 1440-46). Furthermore, there is doubt as to whether anticoagulation is useful for preventing IBMIR. It has been found that, in xenotransplant models, the use of complement depletion or anticoagulation was insufficient to prevent IBMIR (Rood et al. 2007 Transplantation 83:202-210). Cabric, et al. (2006) Cell Transpl 15:759-67 and (2007) Diabetes 56:2008-15) suggest that gene therapy approaches are not appropriate for avoiding IBMIR in pancreatic islets because they introduce new DNA into islets and are associated with a risk of inducing inflammatory or even adaptive immune responses, and transduced islets showed an impaired glucose-stimulated insulin release. They instead suggest pretreatment of islet cells with agents such as heparin.
Although xenotransplantation of islets, particularly from porcine donors, is an appealing alternative to the use of allografts because of the limited supply and quality of human pancreatic islets, major obstacles remain. Both immediate and delayed immune responses and islet destruction require potentially toxic cocktails of immunosuppressant therapies. The production of genetically modified animals to address certain immune responses has been suggested, however this production has met with limited success because of toxicity associated with expression of immunosuppressant in situ. There remains a need for improved animals and tissues suitable for xenotransplantation therapies. In particular, there remains a need for improved animals and tissues to produce insulin producing xenografts that will reduce diabetes in a patient without requiring significant or long term immunosuppressive therapies.